Multi-biomarker biosensor

Abstract

Nanosubstrates as biosensors, methods of making such nanosubstrates, and methods of using such nanosubstrates to detect biomarkers are described.

The invention claimed is: 
     
       1. A nanosubstrate comprising:
 a substrate layer comprising a photoresist material; 
 a conductive layer deposited on the substrate layer; 
 an insulating layer deposited on the conductive layer, the insulating layer interrupted by two or more nanotrenches or nanowells having different widths, the widths being at least about 20 nm; and 
 one or more nanoparticles deposited in each of the two or more nanotrenches or nanowells, the nanoparticles belonging to two or more different size classes, wherein the nanoparticles in each size class have a diameter less than or equal to the width of the nanotrench or nanowell in which they are deposited, and wherein the nanoparticles of each size class are covalently bound to a different antibody or antigen binding fragment thereof. 
 
     
     
       2. The nanosubstrate of  claim 1 , wherein the nanosubstrate comprises an area of about 0.01 mm 2  to about 0.1 mm 2 . 
     
     
       3. The nanosubstrate of  claim 1 , wherein the conductive layer comprises an organic or inorganic conductor. 
     
     
       4. The nanosubstrate of  claim 3 , wherein the conductive layer comprises gold, aluminum, copper, polyanaline, or a combination thereof. 
     
     
       5. The nanosubstrate of  claim 1 , wherein the insulating layer comprises a photoresist material. 
     
     
       6. The nanosubstrate of  claim 5 , wherein the photoresist material is SU-8. 
     
     
       7. The nanosubstrate of  claim 1 , wherein the thickness of the conductive layer is about 40 nm to about 100 nm. 
     
     
       8. The nanosubstrate of  claim 1 , wherein the thickness of the insulating layer is about 80 nm to about 150 nm. 
     
     
       9. A catheter comprising the nanosubstrate of  claim 1 . 
     
     
       10. The nanosubstrate of  claim 1 , wherein the deposited nanoparticles are electrophoretically deposited. 
     
     
       11. The nanosubstrate of  claim 1 , further comprising an adhesion layer beneath said substrate and an additional substrate beneath said adhesion layer. 
     
     
       12. The nanosubstrate of  claim 11 , wherein said additional substrate comprises silicon. 
     
     
       13. The nanosubstrate of  claim 1 , wherein the nanosubstrate comprises a biosensor active area containing said nanotrenches or nanowells and an electrode extension devoid of nanotrenches or nanowells. 
     
     
       14. The nanosubstrate of  claim 13 , wherein the electrode extension comprises a plurality of nanowires, each nanowire formed from an extension of the conductive layer at the base of a single nanotrench or nanowell. 
     
     
       15. The nanosubstrate of  claim 13 , wherein the biosensor active area is suitable for being broken off from the electrode extension. 
     
     
       16. The nanosubstrate of -further comprising an antigen bound to said antibody or antigen binding fragment. 
     
     
       17. The nanosubstrate of  claim 16  further comprising a detection antibody bound to said antigen. 
     
     
       18. The nanosubstrate of  claim 17 , wherein the detection antibody is fluorescently labeled. 
     
     
       19. The nanosubstrate of  claim 18  comprising a plurality of bound antigens and a plurality of bound detection antibodies. 
     
     
       20. The nanosubstrate of  claim 19 , wherein the position of fluorescence on said nanosubstrate indicates the type of antigen bound to the nanosubstrate. 
     
     
       21. The nanosubstrate of  claim 1  that can detect 100 biomarkers. 
     
     
       22. The nanosubstrate of  claim 21  that can detect 1000 biomarkers. 
     
     
       23. The nanosubstrate of  claim 1  that can detect a biomarker diagnostic for a hyperproliferative, hyperplastic, metaplastic, dysplastic, or pre-neoplastic disease or disorder or for a cancerous condition. 
     
     
       24. The catheter of  claim 9  that is inserted into the body of a subject. 
     
     
       25. The catheter of  claim 9  that is contacted with a biological sample of a subject. 
     
     
       26. The nanosubstrate of  claim 1  having a plurality of said nanotrenches or nanowells arranged in an array. 
     
     
       27. The nanosubstrate of  claim 1 , wherein the insulating layer comprises poly(methyl methacrylate).